This P20 application aims to establish an advanced imaging probes program by integrating the biomedical expertise of Emory University (Atlanta, GA), the engineering strength of Georgia Tech (Atlanta, GA), the powerful organic chemistry of Scripps Research Institute (La Jolla, CA), and the single-molecule biophysical innovations of Harvard University (Cambridge, MA). Its broad and long-term goal is to develop a new class of bioconjugated luminescent nanoparticles with dual cellular delivery and targeting functions for real-time and multicolor fluorescence imaging of single-molecule processes in living cells. The proposed research on nanoparticle synthesis, cellular delivery, and organic chemistry is broadly applicable to many types of nanometer-sized particles such as colloidal metal nanoparticles, dye-doped silica, and polymeric nanobeads. But a particular focus will be placed on core-shell semiconductor quantum dots (QDs) because of their novel optical properties such as improved brightness, resistance against photobleaching, and simultaneous multicolor excitation. Quantum dots are also in an intermediate or "mesoscopic" size range (1-10 nm diameter) that provides enough surface area for linking to multiple delivery and targeting ligands while avoiding major kinetic or steric hindrance problems. These properties are beyond the intrinsic capabilities of organic dyes and fluorescent proteins, and are most promising for improving the sensitivity of molecular and cellular imaging by a factor of 10 to 100. In parallel with probe development, we will explore innovative single-molecule imaging and signal processing methods in order to discriminate bound targets from unbound probes inside living cells. In collaboration with cell biologists, the QD probes and single-molecule imaging will be used to study complex molecular events involved in programmed cell death, especially the subcellular localization of p53 protein, nuclear factor kappa B, and androgen receptor, as well as the involvement of microtubules and molecular motors in transporting gene-regulatory proteins from the cytoplasm to the cell nucleus. The proposed development of bioaffinity QD probes should bring major changes to single-molecule biophysics and molecular/cellular biology. Its potential practical outcomes include a new generation of semiconductor QD probes with improved optical properties, small-molecule libraries and biomolecular engineering methods for cellular delivery and targeting of diagnostic and therapeutic agents, and single-molecule imaging hardware and software for use by the broad scientific community.